Calcium Influx, But Not Intracellular Calcium Release, Supports PACAP-Mediated ERK Activation in HEK PAC1 Receptor Cells

Abstract

In HEK cells expressing GFP-tagged PAC1Hop1 receptors, PACAP augments ERK phosphorylation through two parallel pathways; one through PACAP/PAC1 receptor internalization/endosome MEK/ERK signaling, the other through PLC/DAG/PKC activation. We examined whether elevation of intracellular calcium ([Ca2+]i) was required for either of the PACAP/PAC1 receptor-mediated ERK activation mechanisms. The PACAP (25 nM)-induced elevation of [Ca2+]i was greater with cells maintained in Ca2+-containing than in Ca2+-deficient solution, suggesting that both calcium release from internal stores and calcium influx contributed to the rise in [Ca2+]i. A thapsigargin-induced increase in [Ca2+]i also was greater with calcium in the external solution. OAG, the cell permeable analogue of DAG, increased [Ca2+]i, but only in Ca2+-containing solution. Decreasing external calcium or depleting internal calcium stores did not block PACAP-induced PAC1 receptor internalization. Omission of calcium from the external solution, but not thapsigargin pretreatment, significantly blunted PACAP-stimulated ERK phosphorylation. The PKC inhibitor BimI decreased PACAP-mediated ERK activation in both Ca2+-containing or Ca2+-deficient solutions. In contrast, following Pitstop 2 pretreatment to block endocytic mechanisms, PACAP activated ERK only when calcium was present in the external solution. We conclude that the endosome signaling pathway is largely calcium-independent whereas calcium influx appears necessary for the PLC/DAG/PKC component of PACAP-induced ERK activation.

Role of transglutaminase 2 in PAC1 receptor mediated protection against hypoxia-induced cell death and neurite outgrowth in differentiating N2a neuroblastoma cells

“…receptor-induced ERK1/2 is dependent upon calcium influx and PLC/DAG/PKC (May et al, 2014) and hence the inhibition of PACAP-27-induced TG2 by removal of extracellular Ca 2+ and inhibition of PKC may reflect their up-stream role(s) in ERK1/2 activation. In the current study, PAC1 receptor-induced ERK1/2 activation is independent of Ca 2+ influx and partially sensitive to PKC inhibition.…”

PACAP and VIP signaling in chondrogenesis and osteogenesis

“…2). Ca 2+ influx can be evoked by PACAP [90] and the presence of PACAP and VIP is able to decrease the Ca 2+ entry via L-and N-type calcium channels in neurons [91]. It is known that the administration of PACAP affects Ca 2+ oscillation [92] and alters the Ca 2+ related vesicular transport of chromaffin cells [93].…”

Regulation of Intracellular Calcium by Carbon Monoxide in Human Bronchial Epithelial Cells

“…1 E ). Pyr6 only partially suppressed the calcium influx, and the rest may be due to other calcium entry pathways, such as PLC/diacylglycerol (DAG) activated calcium influx [40, 41]. …”